The purpose of this proposal is to elucidate the function of GPRC6A. We have discovered that GPRC6A, an orphan receptor with homology to the calcium (Ca)-sensing receptor CASR, also senses extracellular Ca and is expressed in osteoblasts as well as in the kidney and other tissues. We have found that GPRC6A regulates bone mass and renal calcium excretion, as evidenced by the presence of osteopenia and hypercalciuria in GPRC6A null mice. We also have evidence for a primary defect in osteoblast function in GPRC6A null mice, which exhibit impaired osteoblast-mediated bone formation in vivo. Also, osteoblasts derived from GPRC6A null calvaria show an attenuation of extracellular Ca- and amino acid-sensing properties ex vivo. These findings are the basis of our hypothesis that GPRC6A is a Ca- and amino acid-sensing receptor and is directly and/or indirectly involved in regulating osteoblast-mediated bone formation and renal calcium homeostasis. We have used our hypothesis to make predictions that will be tested by specific experiments. Specific Aim 1 is to complete characterization of the bone phenotype in GPRC6A null mice. We will confirm the presence and investigate the mechanisms of osteopenia in GPRC6A null mice. We will also establish the role of GPRC6A in regulating osteoblast signaling and differentiation ex vivo by comparing the Ca, calcimimetic, and amino acid-sensing properties and maturation potential of cultured osteoblasts derived from GPRC6A-deficient and wild-type mice. Site-directed mutagenesis in GPRC6A of Ca, calcimimetic and amino acid binding sites conserved with CASR will be performed to confirm the functional significance of conserved residues. In addition, we will assess the role of GPRC6A in osteoblasts in sensing factors released from primary stimulation of osteoclast-mediated bone resorption in vivo by transfer of osteoprotegerin (OPG) deficiency onto the GPRC6A null background. Finally, we will use a Cre-loxP strategy to generate mouse lines with the GRPC6A receptor selectively ablated from osteoblasts to establish the primary function of GPRC6A in bone, and to create a model for later exploration of selective GPRC6A function in other tissues. Specific Aim 2 is to complete characterization of the kidney phenotype and define the mechanisms of hypercalciuria in GPRC6A null mice, as well as investigate the interdependence of the kidney and bone abnormalities. We will also investigate the tissue- and cell-specific expression of GPRC6A and screen for potential abnormalities in other tissues that could impact upon the established bone and kidney phenotype in GPRC6A null mice. These studies will establish the role of GPRC6A in maintenance of bone and calcium homeostasis as well as identify other potential functions of this receptor that could lead to modification of our hypothesis. Successful completion of this project is certain to yield important new knowledge regarding the function of GPRC6A.